This proposal examines the hypothesis that agonist-induced trafficking of G-protein coupled receptors (GPCRs) is required for the initiation and termination of signal transduction, and that defects result in uncontrolled stimulation and disease. The molecular mechanisms and physiological roles of substance P (SP)- induced trafficking of the neurokinin 1 receptor (NKIR) will be examined at the level of the cell and the whole animal. It is important to understand the mechanisms of NK1R signaling in view of the pathophysiological roles of this receptor in neurogenic inflammation, pain and intestinal motility. NK1R regulation will be studied in transfected cell lines, endothelial cells and enteric neurons in short-term culture, and in knockout and transgenic mice. Aim 1 will define the molecular mechanism of SP-stimulated endocytosis and intracellular trafficking of the NK1R. The role of the clathrin adapter p-arrestin, and of dynamin and Rab GTPases in NK1R trafficking will be examined by expression of dominant negative mutants, and by studying neurons from p- arrestin knockout mice. The role of the cytoskeleton and of endosomal acidification and phosphatases will be examined using specific drugs Aim 2 will define the importance of NK1R trafficking for the initiation of signal transduction. The role of p-arrestins, Rabs and the cytoskeleton in NKlR-mediated MAP kinase activation will be determined using reagents developed in Aim 1. These studies will define the role of p-arrestins as molecular scaffolds that recruit and organize components of the MAPK cascade. Aim 3 will define the importance of NK1R trafficking in desensitization and resensitization of signaling. The role of G-protein receptor kinases (GRKs) and p-arrestins in desensitization will be determined by expression of dominant negative mutants and by studying neurons from knockout mice. The importance of Rabs, the cytoskeleton and endosomal sorting for resensitization will be examined using reagents from Aim 1. Aim 4 will determine whether defects in mechanisms of NK1R desensilization result in prolonged SP signaling and disease. SP signaling will be examined in mice deficient in GRKs or p-arrestins, or expressing a desensitization and internalization-defective mutant NK1R (NK1R8325, a naturally occurring NK1R variant). The effects of SP on intestinal motility and neurogenic inflammation will be examined, with the expectation that defects in desensitization will result in exaggerated motor and inflammatory responses. Together, the results of these experiments will provide new information about GPCR signaling, and will define how defects in signal transduction can cause disease. PERFORMANCE SFTEfS) (organization, city, state) University of California, San Francisco Harvard University KEY PERSONNEL. SeeInstructions on Page 11. Use continuationpagesasneededtoprovidetherequired Informationin theformat shown below. Name Organization Role on Project Nigel Bunnett, Ph.D. University of California, San Francisco Principal Investigator Eileen Grady, Ph.D. University of California, San Francisco Co-investigator Kimberley Kirkwood, M.D. University of California, San Francisco Co-investigator Fabien Schmidlin, Ph.D. University of California, San Francisco Post-doctoral fellow Norma Gerard, Ph.D. Harvard University Principal Investigator Shoji Okinaga, M.D., Ph. D. Harvard University Post-doctoral Fellow Craig Gerard, M.D. Harvard University Consultant Stephen Collins, M.D. McMaster University Consultant Helen Raybould, Ph.D. University of California, Los Angeles Consultant Jon Seidman, Ph.D. Harvard University Consultant PHS 398 (Rev. 4/98) Page 2 BB Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b.. CC Principal Investigator/Program Director (Last, first, middle) Bunnett, Nigel W., Ph.D. Type the name of the principal investlgatoir/proaju^Tm) director at the top of each printed page and each ooaojftrauation page. (For type specifications, see Specific Instructions onpage 10.) RESEARCH GRANT TABLE OF CONTENTS PAGE NUMBERS Face page 1 Description,